Split-signal FISH for detection of chromosome aberrations in acute lymphoblastic leukemia.

Chromosome aberrations are frequently observed in precursor-B-acute lymphoblastic leukemias (ALL) and T-cell acute lymphoblastic leukemias (T-ALL). These translocations can form leukemia-specific chimeric fusion proteins or they can deregulate expression of an (onco)gene, resulting in aberrant expression or overexpression. Detection of chromosome aberrations is an important tool for risk classification. We developed rapid and sensitive split-signal fluorescent in situ hybridization (FISH) assays for six of the most frequent chromosome aberrations in precursor-B-ALL and T-ALL. The split-signal FISH approach uses two differentially labeled probes, located in one gene at opposite sites of the breakpoint region. Probe sets were developed for the genes TCF3 (E2A) at 19p13, MLL at 11q23, ETV6 at 12p13, BCR at 22q11, SIL-TAL1 at 1q32 and TLX3 (HOX11L2) at 5q35. In normal karyotypes, two colocalized green/red signals are visible, but a translocation results in a split of one of the colocalized signals. Split-signal FISH has three main advantages over the classical fusion-signal FISH approach, which uses two labeled probes located in two genes. First, the detection of a chromosome aberration is independent of the involved partner gene. Second, split-signal FISH allows the identification of the partner gene or chromosome region if metaphase spreads are present, and finally it reduces false-positivity.

t(7;12)(q36;p13) and t(7;12)(q32;p13)--translocations involving ETV6 in children 18 months of age or younger with myeloid disorders.

Our retrospective karyotype review revealed two rare recurrent translocations affecting ETV6 (TEL): t(7;12)(q36;p13) and t(7;12)(q32;p13). Five patients with a t(7;12) were from a group of 125 successfully karyotyped pediatric patients enrolled in consecutive clinical AML trials of the Dutch Childhood Leukemia Study Group over a period of 7 years. During a search of available cytogenetic databases, we found 7q and 12p abnormalities in two additional Dutch patients and in three participants in Pediatric Oncology Group trials. A del(12p) had been initially identified in four of these patients and re-examination of the original karyograms revealed a t(7;12)(q36;p13) in two instances and a probable t(7;12) in the other two. FISH confirmed the presence of a t(7;12)(q36;p13) in the latter. Most (n = 7) also had trisomy 19. The t(7;12)(q36;p13) (n = 9) was more common than the t(7;12)(q32;p13) (n = 1). These subtle translocations were found only in children 18 months of age or younger. A literature search revealed that the t(7;12) with break-points at 7q31-q36 and 12p12-p13 had been reported in six children with myeloid disorders and in two with acute lymphoblastic leukemia; all were 12 months of age or younger. Only two of the 17 for whom survival data were available, were alive after at least 22 months of continuous complete remission. Our findings suggest that ETV6 rearrangements due to a t(7;12) may play an adverse role in myeloid disorders in children 18 months of age or younger. Therefore, children in this age group with myeloid disorders should be screened for both MLL and ETV6 rearrangements.

MDR1 expression in poor-risk acute myeloid leukemia with partial or complete monosomy 7.

Expression of the multidrug resistance (MDR1) phenotype, encoded by the MDR1 gene, is an adverse prognostic factor for CR and survival in acute myeloid leukemia (AML). Other prognostic factors, such as specific cytogenetic abnormalities, have been identified in AML. We have investigated the expression of the MDR1 gene in untreated AML patients with monosomy 7 (n = 12), and partial deletions (n = 7) of the long arm of chromosome 7 (respectively -7/7q-), because of the extremely bad prognosis associated with these cytogenetic abnormalities and because of the fact that the MDR1 gene is located on chromosome 7q21.1. The findings were compared with the level of MDR1 expression in a group of 42 other AML patients, matched for age with favourable, neutral or complex cytogenetic abberations. MDR1 mRNA expression, as measured by the RNase protection assay was significantly higher in the -7/7q- group vs other AML patients (median 1.3 vs 0.1 arbitrary units, P = 0.02). Protein expression of MDR1 in the -7/7q- group, as determined with the monoclonal antibody MRK16, was found to be similar to the levels found in the control group. With a functional rhodamine retention assay using the modulator PSC833, increased MDR1 activity was observed in the -7/7q- group as compared to the control group of patients (P = 0.05). Considering the higher MDR1 mRNA expression and equal or slightly elevated level of protein expression of MDR1, we studied the presence of MDR1 genes in this group of -7/7q- patients. Fluorescence in situ hybridization (FISH) studies, using a specific MDR1 probe revealed no loss of an MDR1 allele in any of the deleted q- arms of the seven patients with 7q-, whereas all monosomy 7 patients lacked one MDR1 gene homologue. To determine whether there was selective loss of the MDR1 gene in the -7/7q- patients, the genetic polymorphism of the MDR1 gene was used. Both allelic variants (G and T) were represented in the -7/7q- and in the control group, showing a predominance for GT at position 2677 of the MDR1 gene in the control group. In the 12 monosomy 7 patients loss of the MDR1 allele was random. Methylation studies of the CpG island of the MDR1 gene revealed no hypermethylation in any of the -7/7q- patients. We conclude that MDR1 expression in -7/7q- AML patients is upregulated at transcriptional, but not at translational level, suggesting that mechanisms other than MDR1 are responsible for the poor prognosis in these patients.

Characterization of mRAD18Sc, a mouse homolog of the yeast postreplication repair gene RAD18.

The RAD18 gene of the yeast Saccharomyces cerevisiae encodes a protein with ssDNA binding activity that interacts with the ubiquitin-conjugating enzyme RAD6 and plays an important role in postreplication repair. We identified and characterized the putative mouse homolog of RAD18, designated mRAD18Sc. The mRAD18Sc open reading frame encodes a 509-amino-acid polypeptide that is strongly conserved in size and sequence between yeast and mammals, with specific conservation of the RING-zinc-finger and the classic zinc-finger domain. The degree of sequence conservation between mRAD18Sc, RAD18, and homologous sequences identified in other species (NuvA from Aspergillus nidulans and Uvs-2 from Neurospora crassa) is entirely consistent with the evolutionary relationship of these organisms, strongly arguing that these genes are one another's homologs. Consistent with the presence of a nuclear translocation signal in the amino acid sequence, we observed the nuclear localization of GFP-tagged mRAD18Sc after stable transfection to HeLa cells. mRNA expression of mRAD18Sc in the mouse was observed in thymus, spleen, brain, and ovary, but was most pronounced in testis, with the highest level of expression in pachytene-stage primary spermatocytes, suggesting that mRAD18Sc plays a role in meiosis of spermatogenesis. Finally, we mapped the mRAD18Sc gene on mouse chromosome 6F.

Maternal stress during hospitalization of the adopted child.

PURPOSE: To identify and describe the experiences of mothers whose adopted children are hospitalized and to compare those experiences to those of mothers whose biological children are hospitalized. DESIGN: Comparative descriptive design. METHODS: Mothers of hospitalized children (n = 33 adopted; n = 19 biological) completed a slightly revised version of the Parental Stressor Scale: Pediatric Intensive Scale Unit (PSS:PICU). Adoptive mothers also completed a questionnaire related to their perceptions of the impact of their child's adoption on the hospitalization experience. RESULTS: Adoptive mothers perceived statistically significantly higher levels of stress related to their child's behavioral/emotional response to hospitalization. Children who had been with the family for less time perceived the hospitalization as more stressful. When both groups of mothers were considered, mothers of younger children perceived a higher level of stress. A majority of adoptive mothers felt the adoption had an impact on the hospitalization experience, especially related to their limited information about their child's medical history, staff lack of knowledge about legal issues, and concern about attachment to the child. CLINICAL IMPLICATIONS: Nurses need to be aware of adoptive mothers' concerns related to attachment issues, limited family medical history, and legal rights in order to provide sensitive and effective care. Inservice education programs could be designed to help teach all staff about these important issues.

Two-colour FISH detection of the inv(16) in interphase nuclei of patients with acute myeloid leukaemia.

The inv(16)(p13q22) and t(16;16)(p13;q22) in acute myeloid leukaemia are associated with a relatively good prognosis but are difficult to detect using classic cytogenetics. We have designed a two-colour fluorescence in situ hybridization approach that uses two DNA probes that map close to and on either side of the inv(16) p-arm breakpoint region. This new strategy clearly detected the inv(16)(p13q22)/t(16;16)(p13;q22) on both metaphase chromosomes and in interphase nuclei, even when they are of poor quality. This procedure also detected the inv(16) in cases with an additional deletion of sequences proximal to the 16p-arm breakpoint which is present in 20% of all cases.

Complete remission of t(11;17) positive acute promyelocytic leukemia induced by all-trans retinoic acid and granulocyte colony-stimulating factor.

The combined use of retinoic acid and chemotherapy has led to an important improvement of cure rates in acute promyelocytic leukemia. Retinoic acid forces terminal maturation of the malignant cells and this application represents the first generally accepted differentiation-based therapy in leukemia. Unfortunately, similar approaches have failed in other types of hematological malignancies suggesting that the applicability is limited to this specific subgroup of patients. This has been endorsed by the notorious lack of response in acute promyelocytic leukemia bearing the variant t(11;17) translocation. Based on the reported synergistic effects of retinoic acid and the hematopoietic growth factor granulocyte colony-stimulating factor (G-CSF), we studied maturation of t(11;17) positive leukemia cells using several combinations of retinoic acid and growth factors. In cultures with retinoic acid or G-CSF the leukemic cells did not differentiate into mature granulocytes, but striking granulocytic differentiation occurred with the combination of both agents. At relapse, the patient was treated with retinoic acid and G-CSF before reinduction chemotherapy. With retinoic acid and G-CSF treatment alone, complete granulocytic maturation of the leukemic cells occurred in vivo, followed by a complete cytogenetical and hematological remission. Bone marrow and blood became negative in fluorescense in situ hybridization analysis and semi-quantitative polymerase chain reaction showed a profound reduction of promyelocytic leukemia zinc finger-retinoic acid receptor-alpha fusion transcripts. This shows that t(11;17) positive leukemia cells are not intrinsically resistant to retinoic acid, provided that the proper costimulus is administered. These observations may encourage the investigation of combinations of all-trans retinoic acid and hematopoietic growth factors in other types of leukemia.

Unique issues of the adopted child: helping parents talk openly and honestly with their child and the community.

1. Due to the psychological impact of losses associated with adoption, adopted children are at risk for developing adjustment problems. Children raised in families that are comfortable talking about adoption are more likely to be emotionally healthy. 2. The child's perception and understanding of adoption changes as the child develops and matures. 3. Health care professionals can teach parents about typical concerns related to adoption and ways to provide information and support consistent with the child's emotional maturity and learning ability. 4. Parents can also learn strategies to counter critical social attitudes and promote a more positive view of adoption.

Cytogenetic clonality analysis: typical patterns in myelodysplastic syndrome and acute myeloid leukaemia.

The cell morphology and karyotype of bone marrow samples from 24 patients with myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) were studied simultaneously with a combined technique of May-Grünwald-Giemsa (MGG) staining and fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes. This enabled us to investigate cell lineage involvement in three malignant conditions: MDS (n = 12), leukaemia-transformed MDS (LT-MDS) (n = 5) and de novo AML (n = 7). In MDS we found blasts and often significant proportions of mature granulocytic and erythroid cells to be cytogenetically abnormal. Percentages of granulocytic and erythroid cells with cytogenetic aberrations were generally less than those of blasts. These data support the involvement of a transformed pluripotent stem cell that has retained maturation abilities. In two patients with chronic myelomonocytic leukaemia (CMMoL) the clonal involvement of monocytes was predominant. Results in the five patients with LT-MDS were similar to those in MDS. In the bone marrow of five of the seven de novo AML patients the cytogenetic abnormalities were restricted to the blasts and did not include the more mature granulocytic or erythroid populations. In the other two patients with AML, both with a t(8;21) and a loss of the Y chromosome, high percentages of mature neutrophils were cytogenetically abnormal. These patterns of clonal lineage involvement in MDS, LT-MDS, t(8;21) AML and AML appear typical and may be of clinical use, for example, for distinguishing LT-MDS from de novo AML in newly presenting patients.

Laryngeal carcinoma after radiation therapy: correlation of abnormal MR imaging signal patterns in laryngeal cartilage with the risk of recurrence.

PURPOSE: To correlate abnormal magnetic resonance (MR) imaging signal patterns in cartilage with the effectiveness of radiation treatment. MATERIALS AND METHODS: Eighty previously untreated patients underwent MR imaging and radiation therapy with a curative intent. Cartilage was considered to have an abnormal signal pattern if it had intermediate signal intensity on T1-weighted spin-echo (SE) MR images and high signal intensity on T2-weighted SE MR images. The minimum follow-up was 2 years. RESULTS: Abnormal MR imaging signal patterns of the thyroid cartilage (P < .001; P < .04) were more ominous than those of other cartilage. Abnormal signal patterns in cartilage of patients with small tumors (< 5 cm3 and especially < 1 cm3) were less significant. Abnormal signal patterns in cartilage combined with a large tumor (> 5 cm3) worsened the prognosis significantly (P < .05). CONCLUSION: Abnormal MR imaging signal patterns in cartilage may not indicate a poor prognosis in every case. Abnormal signal intensity in the thyroid cartilage combined with a tumor volume of > 5 cm3, however, appears to indicate an adverse prognosis with regard to tumor recurrence.

Predictive value of MR imaging-dependent and non-MR imaging-dependent parameters for recurrence of laryngeal cancer after radiation therapy.

PURPOSE: To determine the predictive value of several clinical and radiologic parameters for recurrence of laryngeal cancer. MATERIALS AND METHODS: Eighty previously untreated patients underwent magnetic resonance (MR) imaging before radiation therapy with curative intent. Tumor volume was calculated from T1-weighted MR images. Cartilage was considered invaded by pathologic tissue if it had intermediate signal intensity on T1-weighted spin-echo (SE) MR images and high signal intensity on T2-weighted SE MR images. The minimum follow-up was 2 years. RESULTS: Parameters such as age, sex, histopathologic findings, and invasion of the vocal muscle or pre-epiglottic space were not significantly correlated with tumor recurrence. Logistic regression analysis showed three relevant contributors: cord mobility, as judged clinically, and tumor volume and, more significantly, cartilage invasion, as seen at MR imaging. CONCLUSION: For untreated laryngeal cancer, MR imaging findings of tumor volume and cartilage invasion allow better patient selection for either radiation therapy or surgery. MR imaging is mandatory for T staging of laryngeal cancer.

Hemodynamic effects and image quality of low-osmolar ionic and nonionic contrast media during pulmonary angiography.

RATIONALE AND OBJECTIVES: We compared the hemodynamic responses to ionic and nonionic low-osmolar contrast media of patients who underwent pulmonary angiography. METHODS: Ninety-nine consecutive patients with suspected pulmonary emboli were randomly assigned to receive either 40 ml iohexol or 40 ml ioxaglate in 2 sec at 600 psi (0.17 kg/m2). Mean pulmonary arterial pressure, pulse rate, and blood pressure were recorded before, immediately after, and 2, 5, and 10 min following injection. Image quality was assessed by readers who were unaware of drug assignment. RESULTS: Pulmonary arterial pressure increased to a maximum at 2 min and was higher in patients with pulmonary emboli (p = .06). There were no significant differences between the two contrast media used. The systolic blood pressure and pulse rate in patients with pulmonary emboli increased significantly more in the ioxaglate group (ps = .03 and .04, respectively). Image quality was excellent in 90% of both groups. CONCLUSION: Both contrast agents are safe for pulmonary angiography and yield similar image quality. There appears to be a positive inotropic effect of ioxaglate.

Isolation, properties and chromosomal localization of four closely linked hamster interferon-alpha-encoding genes.

Three recombinant phages containing hamster interferon-alpha-encoding genes (Ha Ifa) were isolated from a Ha genomic library, using a murine (Mu) Ifa probe. The phage inserts contained overlapping genomic fragments which span a total length of approx. 30 kb, on which four Ha Ifa genes are localized. The Ifa gene cluster could be assigned to hamster chromosome 2q. The nt sequences of the four Ifa genes were determined. Two of the genes are functional (Ha Ifa-1 and Ifa-3) and two are pseudogenes (Ifa-ps2 and Ifa-ps4). Ha Ifa-1 and -3 were transiently expressed in COS cells and they gave rise to protein products (A1 and A3, respectively) with antiviral properties on hamster CHO cells. In addition, Ha A1 revealed high antiviral activity on murine L929 cells.

Structure, chromosome localization, and regulation of expression of the interferon-regulated mouse Ifi54/Ifi56 gene family.

The interferon-alpha (IFN-alpha) regulated mouse Ifi54/Ifi56 gene family, which is composed of at least four members (Ifi54, Ifi56, Ifi56-ps1, and Ifi56-ps2), was isolated and characterized. In addition, the chromosomal localization of the four genes was determined. The Ifi54 and Ifi56 genes show an identical organization. Both are composed of a very small first exon and a second exon, which contains the complete open reading frame, except for the ATG start codon and the first two nucleotides of the second codon. In both genes, the two exons are separated by a small intron (5 and 2.5 kb, respectively). Expression of both genes is rapidly induced by IFN-alpha (within 2 h). The Ifi54 promoter region contains two sequences, which are closely related to the interferon stimulated response element (ISRE) consensus sequence (ISRE 1, GGTTTCAATTTCT, and ISRE 2, AGTGTTACTTTCT). The two elements are located directly adjacent to each other. A similar organization was recently established for the hamster Ifi54 promoter (Bluyssen et al., 1994). However, the mouse promoter is 70% less active than the hamster promoter. It turned out that ISRE 2 is hardly active, due to the G at position 4, which is a T in the hamster Ifi54 ISRE 2 and in the ISRE consensus sequence. The Ifi56 promoter region contains at a similar position two functional ISREs of identical strength (ISRE 1, AGTTTCAGTTTCT, and ISRE2, AGTTTCACTTTCC). In the Ifi56 promoter, the two ISRE motifs are separated by 6 bp. In addition to the Ifi56 gene, parts of two closely related genes (Ifi56-ps1 and Ifi56-ps2) were isolated. Both fragments contain an Ifi56-related open reading frame. However, we were unable to isolate the presumed first exon of Ifi56-ps1 and Ifi56-ps2, nor could we show expression of the genes. The Ifi54, Ifi56, Ifi56-ps1, and Ifi56-ps2 genes could all be assigned to mouse chromosome 19D1, suggesting a tight clustering.

Chromosomal localization of three repair genes: the xeroderma pigmentosum group C gene and two human homologs of yeast RAD23.

The nucleotide excision repair (NER) disorder xeroderma pigmentosum (XP) is characterized by sun (UV) sensitivity, predisposition to skin cancer, and extensive genetic heterogeneity. Recently, we reported the cloning and analysis of three human NER genes, XPC, HHR23A, and HHR23B. The previously cloned XPC gene is involved in the common XP complementation group C, which is defective in excision repair of non-transcribed sequences in the genome. The XPC protein was found to be complexed with the product of HHR23B, one of the two human homologs of the Saccharomyces cerevisiae NER gene RAD23. Here we present the chromosomal localization by in situ hybridization using haptenized probes of all three genes. The HHR23A gene was assigned to chromosome 19p13.2. Interestingly, the HHR23B and XPC genes, the product of which forms a tight complex, were found to colocalize on band 3p25.1. Pulsed-field gel electrophoresis revealed that the HHR23B and XPC genes possibly share a MluI restriction fragment of about 625 kb. Potential involvement of the HHR23 genes in human genetic disorders is discussed.

A new B-cell line showing a complex translocation (8;14;18) and BCL2 rearrangement.

A cell line named ROS-50 (Rotterdam suspension cell line no. 50) has been established from peripheral blood of a 69-year-old male with acute lymphoblastic leukemia (FAB type L3). Among the aberrations, cytogenetic analysis showed the presence of 14q+, 18q-, and two 8q- marker chromosomes. With fluorescence in situ hybridization (FISH) we characterized the chromosomal translocations, t(8;14) and t(14;18), in which the same chromosome 14 is involved. PCR analysis demonstrated the presence of on IGH-BCL2 rearrangement with a breakpoint in the minor cluster region (mcr) confirming the t(14;18) characteristic for follicular lymphoma. Additional studies showed high expression of BCL2 protein, an early B-cell immunophenotype, and an unusual pattern of IGH gene rearrangement.

In situ hybridization on May-Grünwald Giemsa-stained bone marrow and blood smears of patients with hematologic disorders allows detection of cell-lineage-specific cytogenetic abnormalities.

Bone marrow and blood from patients with acute myeloid leukemia and myelodysplastic syndrome were studied by simultaneous analysis of cell morphology and karyotype. A combined technique of May-Grünwald Giemsa (MGG) for cell morphology and fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes for detection of cytogenetic aberrations allowed us to investigate cell-lineage-specific chromosomal abnormalities. We introduced video recordings to examine large numbers of cells. Briefly, evaluation was first performed on MGG slides, during which cell position and morphology were recorded on an S-VHS recorder. Subsequently, the same slides were used for FISH. This resulted in the identification of MGG-stained cells on the video screen and, at the same time, the interpretation of FISH signals in the fluorescence microscope. Specimens of bone marrow or blood samples from four patients with different hematologic malignancies were studied. One of these patients was studied before and after cytotoxic treatment. The gain or loss of chromosomes could be detected easily and morphologically assigned to the blasts in all patients and to a variable proportion of the myelomonocytic lineage in two patients, but not to the lymphocytes. Thus, this method provides new possibilities for investigating the clonality of hematologic malignancies.